The invention relates to stable self-replicating episomal expression vectors for expressing a gene of interest in a host cell.
The expression of a foreign gene in a host cell is generally achieved by transferring the gene into the host cell using a gene transfer vector. Gene transfer vectors are available in the art and include for example retrovirus vectors, adenoviral vectors and adenoassociated viral vectors. Many transfer vectors operate by integrating at least the transferred gene, if not the complete gene transfer vector, into the host cell chromosome, although non-integrating transfer vectors are known in the art. The efficiency of stable integration of transfected gene constructs is generally very inefficient (1: 103-106). The disadvantage of using viral vector-based gene transfer is that the amount of genetic material that the vector is able to accommodate is limited by the genetic packaging limitations of the virus. Gene transfer vectors which include large fragments of inserted genetic material are difficult to produce at a sufficiently high titre to be of practical value. Therefore, it is difficult to include additional genetic material in a virus-based vector, for example, gene regulatory elements, without deleteriously affecting stable gene transfer.
Locus Control Regions (LCRs) (Grosveld et al., Cell 51:975-985 (1987)), also known as Dominant Activator Sequences, Locus Activating Regions or Dominant Control Regions, confer tissue-specific, integration-site independent copy number-dependent expression on a linked gene that has been integrated into the chromosome of a host cell. LCRs were originally discovered in the human globin gene system, which exhibited strong position effects when integrated into a chromosome of a host cell in a tissue of a transgenic mouse or a mouse erythro-leukaemia (MEL) cell (see, for example, Magram et al., Nature 315:338-340 (1985); Townes et al., EMBOJ. 4:1715-1723 (1985); Kollias et al., Cell 46:89-94(1986); Antoniou et al., EMBO J. 7:377-384 (1988)). Position effects were overcome when the LCRs were linked directly to such transgenes (Grosveld et al., supra). Other LCRs have since been identified, including the xcex2-globin LCR (xcex2LCR) which promotes gene expression in erythroid tissue and the CD2 LCR which promotes gene expression in T cells (see, for example, Greaves et al., Cell 56:979 (1989), European Patent Application EP-A-0 668 357), themacrophage-specific lysozyme LCR (Boniferet al., (1985, 1990)), and a class II MHC LCR (Carson et al., Nucleic Acids Res. 21,9:2065-2072 (1993)).
The present invention provides a stable gene transfer system which, when present in a host cell, confers stable and tissue-restricted expression of a gene of interest carried in the vector.
This invention provides self-replicating, LCR-containing, episomal expression vectors into which a gene of interest is inserted for expression of the gene in cells of a specific tissue-type.
The invention therefore encompasses a self-replicating episomal DNA expression vector for expressing a gene of interest in a host cell in a tissue-restricted manner, the vector comprising: (a) an origin of replication capable of directing replication of the DNA expression vector in cells of the specific type of tissue; and (b) an LCR, or component thereof, which when operatively linked to a gene of interest and present in a host cell directs expression of the gene in a tissue-restricted manner.
A vector according to the invention also may include the gene of interest inserted into a cloning site and operatively linked to the LCR.
The term gene is used to define any DNA sequence capable of being expressed. The gene of interest may be a foreign or heterologous gene, that is, a gene that is either not normally found in the genomic DNA of the host cell or is not normally expressed in that host cell. The gene of interest also may be an artificial DNA sequence.
The tissue-restricted expression of a gene of interest is mediated in vectors of the invention using an appropriate LCR, or components or portions thereof, which confer tissue-type specific expression on the gene of interest.
As used herein, a locus control region (LCR) is defined as a genetic element which is obtained from a tissue-specific locus of a eukaryotic host cell and which, when linked to a gene of interest and integrated into a chromosome of a host cell, confers tissue-specific, integration-site independent (position independent), copy number-dependent expression on the gene of interest. An LCR that is usefil according to the invention possesses these characteristics when integrated into chromosomal DNA, and will retain the ability to confer tissue-type restricted expression of a linked gene when present in a self-replicating episomal vector according to the invention. A component of an LCR refers to a portion of an LCR that also confers tissue-restricted gene expression when linked to a gene and integrated into a self-replicating episomal vector. An LCR may be identified structurally in that it is associated with one or more DNase I hypersensitive sites in its natural chromosomal context, and a component of an LCR useful according to the invention will also encompass at least one DNase I hypersensitive site.
An enhancer is defined herein as a genetic element which increases the level of transcription of a linked gene when present on a self-replicating episomal vector, but which does not confer tissue-specific gene expression when present on the vector.
It is preferred that the vector comprises a component of an LCR which confers tissue-specific expression and contains at least one DNase I hypersensitive site. In the human xcex2-globin LCR, the preferred component of the LCR consists essentially of HS3; also preferred is the human xcex2-globin LCR excluding site HS2; also preferred are sites HS3 and HS4 together without site HS2.
The invention also encompasses a pair of vectors comprising a self-replicating episomal DNA expression system for expressing a gene of interest in a host cell in a tissue-restricted manner, the pair of vectors comprising: a first vector comprising (a) an origin of replication; (b) an LCR, or component thereof, which when operatively linked to a gene of interest and present in a host cell directs expression of the gene in a tissue-restricted manner; and (c) a cloning site for a gene of interest; and a second vector comprising (d) an origin of replication; and (e) a sequence encoding a replication protein, the replication protein being necessary for replication of the origin of replication.
In another embodiment, the second vector also may include an LCR, which may be the same LCR as is present on the first vector or may be a different LCR which specifies the same or at least an overlapping tissue specificity as the first LCR such that the gene of interest and the viral replication gene are expressed in some of the same cells.
It is preferred that for red cell-restricted gene expression, the xcex2-globin LCR from the xcex2-globin locus be used. As used herein, red cells refer to cells of erythroid lineage.
It is preferred that for T-cell restricted gene expression, the CD2 LCR from the CD2 locus be used, or a component thereofcontaining at least one DNase I hypersensitive site that directs T-cell restricted gene expression in an episomal context. It is preferred that for class II MHC-bearing cell restricted gene expression the class II MHC LCR be used, or a component thereof containing at least one DNase I hypersensitive site that directs MHC-bearing cell restricted gene expression in an episomal context. It also is preferred that for macrophage cell restricted gene expression the macrophage/lysozyme LCR be used, or a component thereof containing at least one DNase I hypersensitive site that directs macrophage-cell restricted gene expression in an episomal context.
The term episomal vector refers to a nucleic acid vector which may be linear or circular, and which is usually double-stranded in form. A vector according to the invention is generally within the size range of 1 kb-1,0000 kb, the preferred size range being on the order of 5 kb-100 kb.
The terms self-replicating, stably maintained and persistence are defined herein as follows. The self-replicating function of a vector of the invention enables the vectors to be stably maintained in cells, independently of genomic DNA replication, and to persist in progeny cells for three or more cell divisions without a significant loss in copy number of the vector in the cells, i.e., without loss of greater than an average of about 50% of the vector molecules in progeny cells between a given cell division. This self-replicating function is provided by using a viral origin of replication and providing one or more viral replication factors that are required for replication mediated by that particular viral origin. Origins of replication and, if necessary, any replication factors may be used from a variety of viruses, including Epstein-Barr virus (EBV), human and bovine papilloma viruses, and papovavirus BK.
In a preferred embodiment, the viral origin of replication is the oriP of EBV and the replication protein factor is the trans-acting EBNA-1 protein. EBNA-1 may be provided by expression of the EBNA-1 gene on the same episomal expression vector carrying OriP or on another vector in the cell or from an EBNA-1 gene in the genomic DNA of the host cell.
In a preferred embodiment, a gene encoding a viral replication factor may be present on the self-replicating episomal vector, i.e., on either the same vector that carries the gene of interest or on another vector of a pair of vectors, and operatively linked to an LCR.
As used herein, linked refers to a cis-linkage in which the gene of interest and/or the gene encoding a viral replication factor and the LCR are contained in the same vector and thus present in cis on the same DNA, and operatively linked refers to a cis linkage in which the gene of interest and/or viral replication gene is subject to tissue-restricted expression via the LCR.
Optionally, a transcription terminator may be introduced into a vector of the invention, preferably between the LCR, or component thereof, and the promoter of the gene of interest or the gene(s) encoding viral replication protein(s) to prevent undesirable transcription of these gene from other promoters that may be present on the vectors. Alternatively, a transcription terminator may be introduced upstream of the LCR and downstream of the gene of interest or viral replication gene(s).
The episomal expression vectors of the invention may be delivered to cells in vivo, ex vivo, or in vitro by any of a variety of the methods employed to deliver DNA molecules to cells. The vectors may also be delivered alone or in the form of a pharmaceutical composition that enhances delivery to cells in the body.
In a preferred embodiment, the vectors ofthis invention are used in gene therapy to express a therapeutically useful protein in the cells of a specific diseased tissue, including tumor tissue, in the body.
Vectors of the invention also are used to express a therapeutically useful protein in cells of a specific tissue-type in vitro.
In another embodiment of this invention, the vectors described herein are used to produce a type of transgenic animal in which a foreign or heterologous gene is expressed only in a specific tissue type, as directed by the LCR, or component thereof, incorporated into the vector. The self-replicating function of the vector ensures that the vector will be passed on to the progeny of the transgenic animal. Such transgenic animals are particularly useful in testing the fidelity and efficacy of tissue-specific gene expression prior to clinical treatment of humans.
In another embodiment of the present invention there is provided a method for identifying an LCR or component thereof which when comprised in an episomal DNA expression vector, operatively linked to a gene of interest and present in a host cell directs expression of said gene in a tissue-restricted manner, comprising:
i. testing the LCR or component thereof by transfecting an episomal vector containing the candidate LCR or component thereof operatively linked to a marker gene into a cell line in which the LCR when integrated is active and also into a cell line in which the LCR when integrated is inactive; and
ii. identifying the LCR or component thereof which is only active in the cell line in which the LCR when integrated is active.
Further features and advantages of the invention are found in the following drawings, description and claims.